Selenophosphate synthetase (SPS), the selD gene product from Escherichia coli, catalyzes the biosynthesis of monoselenophosphate, AMP, and orthophosphate in a 1:1:1 ratio from selenide and ATP. In vitro characterization of selenophosphate synthetase from E. coli and Haemophilus influezae revealed that the determined Km value for selenide is far above the optimal concentration needed for growth of various bacterial species and approached levels which are considered toxic. Selenocysteine lyase enzymes, which decompose selenocysteine to elemental selenium (Se0) and alanine, were considered as candidates for the control of free selenium levels in vivo. The ability of a lyase protein to generate Se0 in the proximity of SPS may be an attractive solution to the obstacle of selenium toxicity as well as the high Km value for selenide. Three E. coli NifS-like proteins, CsdB, CSD, and IscS, were characterized. All three proteins exhibit lyase activity on L-cysteine and L-selenocysteine as substrates and produce sulfane sulfur, S0, or Se0, respectively. Each lyase can effectively mobilize Se0 from L-selenocysteine for selenophosphate biosynthesis. In fact, the catalytic activity of SPS is increased when measured in an in vitro coupled assay system in which free selenide is replaced with L-selenocysteine and a lyase protein. Evidence in support of the participation of the E. coli NifS-like proteins in selenium mobilization in vivo comes from additional experiments showing a decreased 75Se content in the selenocysteine-containing enzyme formate dehydrogenase (FDHH) as well as in selenouridine of tRNA when E. coli was grown on 75SeO3 in the presence of increasing concentrations of unlabeled L-selenocysteine. Supplementation of L-selenocysteine up to 0.2 mM in cultures containing 0.1 mM 75SeO3 decreased 75Se incorporation into FDHH by 85% and in selenouridine of tRNA by 77%. Since both the conversion of 2-thiouridine to 2-selenouridine and the incorporation of selenocysteine into FDHH are dependent on selenophosphate, the effective dilution of 75Se supports the participation of the NifS-like proteins in providing selenium for selenophosphate biosynthesis. Competition experiments performed between CsdB and SPS did not support the direct transfer of Se0 between the two proteins and opened up the possibility that an additional protein may function as a selenotransferase. Current work is focused on identifying additional proteins and/or cellular components that may participate in the delivery of Se0 to SPS.